Experiment Overview
| Repository ID: | FR-FCM-Z6XR | Experiment name: | Flow cytometry quantification of HPG Click-iT staining in MB135iDUX4 myoblasts | MIFlowCyt score: | 48.50% |
| Primary researcher: | Danielle Hamm | PI/manager: | Danielle Hamm | Uploaded by: | Danielle Hamm |
| Experiment dates: | 2022-10-11 - 2022-10-11 | Dataset uploaded: | Aug 2023 | Last updated: | Aug 2023 |
| Keywords: | [click chemistry] [protein synthesis] [DUX4] | Manuscripts: | |||
| Organizations: |
Fred Hutchinson Cancer Research Center, Seattle, WA (USA)
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| Purpose: | We used metabolic labeling with methionine analog L-homopropargylglycine (HPG) followed by fixation and Click-iT chemistry to measure levels of nascent protein synthesis 0-92h following a pulse of DUX4. | ||||
| Conclusion: | We confirmed DUX4 inhibition of protein synthesis by labeling cells with methionine analog L-homopropargylglycine (HPG) followed by fixation and Click-iT chemistry, wherein fluorescence microscopy and flow cytometry showed a dramatic reduction in HPG-labeled peptides after a pulse of DUX4, but not with transcriptionally inactive DUX4(F67A). | ||||
| Comments: | Cells were incubated for 30 minutes in DMEM media depleted for methionine and cysteine, followed by a 1-hour incubation in methionine-depleted media supplemented with 200µM L-HPG HCl (Sigma-Aldrich Cat#900893). Cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized 0.5% TritonX-100, and stained with Click-iT HPG Alexa Fluor 488 Protein Synthesis Assay Kit (Invitrogen/Thermo Fisher Cat#C10428) according to manufacturer’s protocol. Cells were resuspended in FACS buffer for analysis by flow cytometry using BD LSRFortessa X-50 with BD FACSDiva software. Data were analyzed using FlowJo v10.5.3. | ||||
| Funding: | Not disclosed | ||||
| Quality control: | None | ||||
